This is to certify that the thesis entitled Effects of Allelopathic Substances Produced by Asparagus on the Incidence and Severity of Fusarium Crown Rot presented by Anne Cothran Hartung has been accepted towards fulfillment of the requirements for Wegree in Science MTQW Major professor 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution ! g' 2:? w E, .. P .45, t 3 .-._' 'lo- :} ; (:1 €¢I .- , 1 £‘: 'u' ‘3 V ' - '3. ‘2'. 3;: ..‘u ‘J 6,3 I ““4 r s 2 x 3,-g§.$% ' V v g. .3. K t i‘ g" I,“ 2‘ ' 4 ”1-1.1..1,‘ “La 1 E} «so 6 12'6"- £1.43! \...___ _ ,~__,Jr MSU LIBRARIES “ RETURNING MATEngggz Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. , , v ,. 3,1,: g r, {1 m m: I.“ ‘I I 'L'" , . .- 5-. 5‘ ‘ ' f _ h . . pr .- 9; ",2. ‘35:" J “l p '1"; ".5 fl,“ EFFECTS OF ALLELOPATHIC SUBSTANCES PRODUCED BY ASPARAGUS ON THE INCIDENCE AND SEVERITY OF FUSARIUM CROWN ROT By Anne Cothran Hartung A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1983 ABSTRACT EFFECTS OF ALLELOPATHIC SUBSTANCES PRODUCED BY ASPARAGUS ON THE INCIDENCE AND SEVERITY OF FUSARIUM CROWN ROT By Anne Cothran Hartung The effects of toxic components isolated from asparagus tissue on Fusarium spp. and other microorganisms and their effects on the susceptibility of asparagus to Fusarium crown rot were investigated to determine their role in Asparagus Decline. Asparagus root tissue alone and treatments in which asparagus root and rhizome tissue were combined with Fusarium inoculum significantly reduced plant growth over untreated controls and treatments with Fusarium alone. Extracts of root tissue were partitioned with solvents, eluted on a Sephadex G-25 column and separated by thin layer chromatography. Four separate components inhibitory to cress seed germination were obtained. Water extracts of root tissues were also inhibitory to growth of Pythium ultimum and 15 of 54 bacterial isolates, but not Fusarium isolates. Extracts of asparagus roots were more inhibitory to microorganisms and seed germination than extracts of fern or rhizome tissue. DEDICATION To my beautiful daughter, Chloe, whose presence made this thesis possible. To my best friend and intimate companion, John, for his constant and loving encouragement. To my father, who always wanted this for me. ii AKNOWLEDGEMENTS I wish to express my gratitude and appreciation to my major professor, Dr. Christine Stephens, for her interest and patience with me as a student, her constant encouragement and confidence in my research abilities, and last but not least, her financial support for this research. I would also like to thank the other members of my committee, Dr. Karen Baker, Dr. Alan Putnam and Dr. John Lockwood for their many helpful suggestions and encouragment for my project as well as their critical review of my thesis. I am also deeply indebeted to Dr. Gary Mills for his interest in my work and his kind and patient assistance and encouragement in developing the isolation procedure. I also extend my appreciation to Tom Stebbins for his technical assistance and his unerring good nature, Bob Livingston for many helpful suggestions, and Dr. Don Ramsdell for his encouragement. Finally I wish to thank the students of the Department of Botany and Plant Pathology for their continual emotional support and for providing a stimulating environment for learning and research. iii TABLE OF CONTENTS List of Tables . . . . . . List of Figures . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . Fusarium Rot and Crown Rot of Asparagus . . . . . . . Control of the Fusarium Pathogen . . . . . . . . . . Allelopathy . . . . . . . . . . . . . . . Asparagus as an Allelopathic Plant . . . . . . . . . CHAPTER 1 ASPARAGUS TISSUE AND FUSARIUM SPP. INTERACTION ON THE INCIDENCE AND SEVERITY OF ROOT AND CROWN ROT OF ASPARAGUS Introduction . . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . . . . Results . . . . . Discussion . . . . . . CHAPTER II ISOLATION OF GERMINATION INHIBITORS FROM DRIED ASPARAGUS ROOT TISSUE Introduction . . . . . . . . . . . . . . . . . . . Materials and Methods . . Results . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . CHAPTER III INTERACTION OF ASPARAGUS ROOT EXTRACTS WITH MICROORGANISMS Introduction . . . . . . . . . . . . . . . . . . Materials and Methods . . . . . . . . Results . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . List of References . . . . . . . . . . . . . . . . . . . iv Page vi 21 21 25 31 39 7 ,»9 44 47 SO SO 53 56 58 TABLE 10 LIST OF TABLES CHAPTER I Dry weight and root rot rating for asparagus seedlings treated with combinations of Fusarium spp. and dried asparagus tissue . . Analysis of variance of dry weight of 3 month old asparagus seedlings treated with Fusarium Spp. and dried asparagus root or rhizome tissue Dry weight and root rot rating for asparagus seedlings treated with combinations of Fusarium spp. and dried asparagus fern tissue Dry weight of 3—month old asparagus seedlings treated with varying levels of dried asparagus root tissue alone or in combination with F. oxysporum f. sp. asparagi or F. moniliforfie Fresh weight of 3 month old asparagus seedlings treated with varying levels of dried asparagus root tissue alone or in combination with F. oxysporum f. sp. asparagi or F. moniliforme asparagi or F. monilifOFme. Analysis of variance of fresh weight of 3 month old asparagus seedlings treated with varying levels of dried asparagus root tissue alone or in combination with F. oxysporum f. sp asparagi or F. moniliforme. Analysis of variance of dry weight of 3 month old asparagus seedlings treated with varying levels of dried asparagus root tissue alone or in combination with F. oxysporum f. sp. Root rot rating for asparagus seedlings treated with combinations of Fusarium spp. and varying levels of dried asparagus root tissue CHAPTER II Components isolated from dried asparagus root tissue which inhibit cress seed germination CHAPTER III Weight of Pythium ultimum hyphae grown in PDB in the presence of various concentrations of asparagus root extract PAGE 26 30 32 KN \JJ 35 36 45 55 LIST OF FIGURES FIGURE INTRODUCTION 1 Asparagus plantation with long "skips" in asparagus rows. Dead plants were caused by Fusarium infection of the roots ----------- 2 Mature asparagus fern showing above ground symptoms of Fusarium infection. Fusarium oxysporum f. sp. asparagi was isolated from tHe crown of this plant .. . .. . .. . .. . 3 Bare circular area in an asparagus plantation attributed to Fusarium .. . .. CHAPTER I 4 Root rot of asparagus caused by a combination of F, oxysporum f. sp. sparagi and asparagus dried tissue (crown residue = dried rhizome root residue = dried root tissue) 5 Root rot of asparagus caused by a combination of F, moniliforme and dried asparagus .. . . CHAPTER II 6 Flow chart for extraction procedure of asparagus root tissue. CHAPTER III 7 Pythium ultimum growing in the presence of dried sterilized root, rhizome or fern tissue. 1) fern tissue, 2) root tissue, 3) rhizome tissue . vi PAGE 5 27 27 54 INTRODUCTION Asparagus officinalis L. (asparagus) is a monocotyledonous plant in the Liliaceae first described by Linnaeus in 1735. The genus Asparagus, considered to be a native of Europe and Central Asia, is comprised of BOO species, some herbaceous, and some woody (30). Only A. officinalis var. altilis L. has been utilized as a commercial crOp. A. officinalis var. altilis has been under cultivation for over 2,000 years in Europe and Asia where it was prized as a food by the Greeks and Romans. Pliny mentions an asparagus plant growing near Ravenna of which "three heads would weigh a pound" (18). Throughout other areas of the old world, asparagus was used mainly as a medicinal plant. Asparagus was brought to America by early colonists where it soon escaped cultivation, adapting itself to sandy fields, road sides and surviving in old garden plots and along salt marshes (13.55)- All species of asparagus are perennial and possess fleshy or tuberous roots and an underground stem (rhizome) that supports ther aerial shoots known as the ferns. The ferns contain cladophylls which are the true leaves. The green branches function as the major photosynthetic organs. The fleshy roots function as storage organs for carbohydrates which are produced during the growing season. In the spring, these reserves are used for the initiation of buds, Spear and fern growth (49). The fleshy roots of the cultivated species may spread laterally 10-12 feet in the soil as they grow outward and downward from the rhizome (22). Individual asparagus plants will differ considerably in growth habit due to the dioecious nature of asparagus: plants of the same variety are not isogenic (53). Seeds are produced in a 3-celled berry that turns red at maturity. Seeds are black, comparatively large (1/8 inch diameter) and are flattened on one side (54). Today, A. officinalis var. altilis is grown as a high cash value vegetable crop throughout the United States. In Michigan, the third largest producer of asparagus behind California and Washington, asparagus is grown on sandy soils often not suitable for other vegetable crops. Therefore, asparagus provides growers a profitable alternative crop suitable for these marginal soils. Properly maintained, asparagus plantations should remain pro- ductive for up to 20 years, producing annual yields of 3,000 pounds per acre or more. But despite increased production due to increased acreage, yields of asparagus are currently declining significantly, causing great consternation among asparagus growers. Today, asparagus fields are being removed from production after 8 to 15 years due to sparse stands and small spear size (52). In 1978, the average yield for Michigan was 1,500 pounds per acre whereas in 1981, the state average was 900 pounds per acre (1). Most fields are planted with approximately 10,000 crowns per acre, but fewer than half of the original crowns survive the first five years (Figure 1). In a 1978 field survey of asparagus fields in Michigan, the average crown population was 3,153 crowns per acre (41), representing a 70% reduction in crown survival. This decline in both yield and number of crowns in the field is known as "Asparagus Decline", and is not confined to Michigan. Reduced yield has also led to decline in acreage planted to asparagus in other asparagus producing states. Over the past 25 years, asparagus production in New Jersey has dwindled from 30,000 acres to less than 1,000 acres (20). In California, reports acreage planted to asparagus decreased from 44,000 acres in 1974 to 28,000 acres in 1978 (52). Asparagus Decline has also been reported in the Netherlands where acreage planted to asparagus decreased from 500 ha in 1963 to 340 ha in 1970 (57). In addition to the decreased longevity and productivity of established plantings, asparagus cannot be reestablished in fields where asparagus was previously grown. Growers have tried replanting in the past but with little success. In severe cases, only 55% of asparagus crowns survived when replanted in fields once containing asparagus (41). Hanna (1947) reported that in fields replanted immediately after plowing out an old asparagus stand, yields were never more than half the expected poundage (17). He also reported that when asparagus was direct seeded into old land, seedling mortality was practically 100% after 2-3 months. Fusarium Root and Crown Rot of Asparagus Asparagus Decline is frequently attributed to root and crown rot of the asparagus plant caused by Fusarium species. Fusaria are among the most cosmopolitan of the fungi. They are of great economic importance since they play a major role in reducing yields and quality of many important food crops of the world (37). Many of the "modern" Fusarium diseases such as corn stalk rot are caused by a complex of organisms aided and directed by a plethora of environmental factors and cultural practices (61). The Fusaria are capable of surviving in the soil almost indefinitely as chlamydospores or other resting structures. They also have the ability to infect plants by several different morphological structures, e.g. chlamydospores, macro- and microconidia. The Fusarium species implicated in causing root and crown rot of asparagus are E. moniliforme Sheldon Snyd. and Hans. and F. oxysporum (Schlecht.) Snyd. & Hans. f. Sp. asparagi Cohen. F. oxysporum is the most frequent— ly isolated of the Fusarium species in soils (36); it is very active as a saprophyte. The pathogenic F. oxysporum species are as specialized as the Uredineae in their specificity to host plants and play a definitive role in plant disease. Fusarium moniliforme is distributed throughout the world but is most common in the warmer regions. It is a major parasite on several species of the Gramineae, particularly rice. However its parasitic activity on other plants is not as well defined as other Fusaria. It is often found in association with other organisms, particularly .E‘ oxysporum, acting in consort with them to produce disease (4.19.36)- Fusarium wilt and root rot of asparagus is considered to be the most common disease of asparagus in the United States (59). The above ground symptoms include a yellowing as well as stunting or wilting of the ferns (Figure 2). Eliptical reddish—brown lesions may be found at the base of the ferns near the soil rigure 1. Asparagus plantation with long "skips" in asparagus rows. Dead plants were caused by Fusarium infection of the roots. Iigure 2. Mature asparagus fern showing above ground symptoms of Fusarium infection. Fusarium oxysporum f. sp. separupi was isolated irom the crown of this plant. line and the vascular bundles of the crown and stalks appear discolored. The first report of the Fusarium disease on asparagus was in 1908 by Stone and Chapman in Massachusetts (50). They described a severe wilting of the young shoots and yellowing of the mature stalks of asparagus plants. An unidentified Fusarium species was isolated from young, wilted shoots but they conducted no pathogenicity studies. Cook, in 1923, reported that stunted, yellowed, plants showing vascular discoloration within the stems occurred in circular areas in infected stands (Figure 3) (8). He was able to complete Koch's postulates with a Fusarium species isolated from infected plants but could not identify the particular species responsible for the symptoms. The first comprehensive report on wilt and root rot of asparagus was written by Cohen and Heald in 1941 (7). These researchers reported the presence of wilt and root rot disease in all asparagus fields surveyed. The isolated F. oxysporum from vascular bundles of roots, crowns, and shoots as well as asparagus crown obtained from other areas of the country. In pathogenicity studies, they reported the "incubation period" of the disease was shorter in sandy soils than in soils with a higher amount of organic matter, and disease development was increased in experiments conducted under high temperature con- ditions. The isolate was not pathogenic on tomato, potato, carnations or onions, but caused a mild wilt of Alaska pea. Armstrong & Armstrong (2) later conclusively established that F. oxysporum f. sp. asparagi was very specific in its pathogenicity to asparagus. Cohen and Heald (7) felt the 7 i.yur( '. Lnrv vlrleHr urea in an anpnrufus Plantation attributpd to Fusarium. natural growth habit of the asparagus plant and the cultural practices employed for its maintenance were contributing factors in the pathogen's etiology. The perennial asparagus storage roots are continually replenished leaving senescing, depleated tissue in the soil that can be easily colonized by the fungus saprophytically. Moreover, since asparagus is harvested by cutting or snapping the spears from the crown, thereby producing a wound, the pathogen is able to easily infect its host. Cohen and Heald concluded that fertilization and pH adjustments of the soil would have little effect in controlling the disease. Field plots for testing fertilizers and soils of pH ranging from 6 to 8.4 showed no differences in percentage of infected plants. They felt the organism was omnipresent wherever asparagus was grown and was specific for asparagus in pathogenicity. In 1955, K.M. Graham published an extensive report concerning infection by Fusarium species on asparagus seedlings (14). He identified the pathogenic organism as F. oxysporum var. redolens (Wr.), and gave a detailed description of how the pathogen invades the asparagus seedling tissue. Sectioning roots from greenhouse-grown inoculated plants, he found the fungus capable of invading the meristematic region of the root tip and through the stomata of the hypocotyl and the coleoptile. Penetration was always initiated intercellularly. By examining many seedlings with many sites of infection, he concluded that most of the infections occurred in the region of the root tip. His examination of mature crowns showed that discolored vascular tissue was most frequently associated with a wound, leading him to suggest this pathogen was a "wound parasite". Endo later established that asparagus storage roots quickly "wall off" Fusarium infections and wounds which are potential infection sites. Stressed storage roots take longer to form wound periderm than non-stressed storage roots, which may give the pathogen an opportunity to cause further infection (Endo, personal communication). Graham also became curious about the frequency of repeated isolation of F. moniliforme from seeds and dead asparagus stalks, and suspected that F. moniliforme might also be affecting seedlings. Although this fungus had been previously considered a saprophyte it caused distinct necrosis of the root apical and lateral meristems of seedlings in greenhouse tests. He did not feel this organism was responsible for the seedling blight observed in the field since symptoms on these plants were dissimilar. Yet he was skeptical of the saprophytic guise attributed to F. moniliforme and cautioned against ignoring the role this fungus may play in the asparagus ecosystem. He compared isolates of both Fusarium organisms in liquid culture and found his isolate 0f.§° oxysporum grew more profusely on sucrose, less on basal medium plus cellulose, and had a greater ability to hydrolyse starch than F. moniliforme. As an aside, he noted F. moniliforme was incapable of producing chlamydospores at even the lowest concentrations of sugar. Graham also examined temperature and moisture effects in greenhouse studies utilizing naturally and artificially infested soils. He placed the optimum soil temperature for the disease 10 between 25—3000 and indicated that low and high soil moisture delayed seedling emergence, favoring pre-emergence damping off. His data from naturally and artificially Fusarium-infested soil indicated that there was a greater than 50% decrease in emergence for all levels of moisture. It was not clear whether he was using surface sterilized seeds in his experiments and this fact reduces the value of his data inasmuch as the pathogen was later shown to be seed borne (12,15,16,21). By the 1950's, it was well accepted among growers throughout the country that asparagus could not be replanted into old fields and virgin soil must be utilized for producing seedlings. By that time, growers were running out of new land. Also, their fields were reaching peak production after 8 years then declining steadily until stands could no longer be profitably maintained. Some asparagus fields would begin to decline after only 4 or 5 years of growth (52). Since asparagus could not be harvested until the third year of growth in a plantation, maximum production of the crop was only lasting 5 to 13 years, based on previous experience, growers expected fields to remain profitable for at least 17 years. Grogan and Kimble responded to this concern and further investigated the Asparagus Decline syndrome in California (14). In the end, they concluded that the problem was the same Fusarium wilt that Cohen and Heald had described, but the strain in California was more virulent than that in Washington. They called their isolate F. oxysporum f. asparagi. They agreed with Kimble that F. moniliforme was also attributing to the problem but F. oxysporum f. asparagi was 11 the primary pathogen responsible for Asparagus Decline. Although their isolate was virulent enough to quickly deplete and kill asparagus plants, the movement of the pathogen in the soil was slow. They also tested their isolate for pathogenicity to other possible hosts but found the pathogen highly specific for asparagus, readily infecting both seedlings and three-month— old crowns. They demonstrated that the pathogen could be seed- borne by washing asparagus seed with water, pouring the wash water over surface sterilized seed, planting the seed in sterile soil, and determining wilt of emerged seedlings. The Fusarium pathogen could be isolated from all seedlings that wilted. They postulated the Fusarium pathogen was spread throughout the field by cultivation when an old stand was removed. This dissemination of the pathogen was responsible for the increased seedling death death noted in the replanted land. They suggested resistant varieties were the only viable control and began the process of developing resistant clones. Lewis and Shoemaker began a screening process for resistant plants and found one line of plants that exhibited limited tolerance to Fusarium (31,32). Takatori, in California, and Ellison, in New Jersey, have also worked on breeding asparagus but to date no cultivars that are resistant to Fusarium have been developed (52). Cultivars such as UC 157 may show tolerance to Fusarium in one area of the country but not in others (Stephens, C.T., personal communication). Although F. moniliforme was a recognized pathogen of asparagus and had been reported as a seed contaminant, it was 12 not considered important in the decline syndrome until 1979 (11,23). Johnson, Springer, and Lewis found F. moniliforme to be the dominant Fusarium species isolated from a 12 year old field (23). They conducted pathogenicity tests to compare the effects of two isolates of F. moniliforme and two isolates of .E' oxysporum f. sp. asparagi on two cultivars of asparagus seedlings and crown. .3' moniliforme was recovered more frequently from stem and crown lesions of plants in 12-year— old plantings whereas F. oxysporum f. sp. asparagi was isolated more frequently from discolored vascular root tissue and cortical root tissue in 2-year-old plantings. They felt F. moniliforme was more important in older plantings of asparagus and proposed the disease this pathogen caused on asparagus be named "Fusarium stem and crown rot." In passing, they mentioned their seedling transplants showed reduced vigor within 3 days of transplanting into Fusarium- infested soil. They suggested this may not be due to the Fusarium infection but to the presence of a "toxin" in the soil since plants exhibited foliar chlorosis and necrosis within a few days of transplanting. They suggested this toxin was being produced by fungal spore germination. Other researchers have studied in detail toxic compounds from Fusarium species. Many different metabolites termed "mycotoxins" have been isolated from Fusarium species in the past decade (58). However, very little evidence has ever been accumulated in support of the hypothesis that such toxins contribute to disease deveIOpment of Fusarium pathogens in any way (33). Control of the Fusarium Pathogen Efforts to develop a chemical control for the Fusarium pathogens have not been successful. Soil fumigation and seed treatment reduce but do not eliminate decline symptoms in the field. Manning (33) reported soil fumigation and preplant crown soaks with benomyl or captafol may increase the average fresh weight of ferns when compared to non-treated plants and non- fumigated soils. However, this combination of treatments, although effective in helping establish newly planted fields does nothing to stop the demise of long term effects of Fusarium infestation in a plantation (28). More recently, soaking seed in 25,000 ppm benomyl in acetone for 24 hrs was found to eradicate Fusarium inoculum from seed (9). However, no data were given as to how eradication of Fusarium from seed may affect the incidence or severity of infestation in plantations as they continue to age. To date, there is no practical chemical control for the pathogens, nor are there any resistant cultivars available. Control is confounded by the perennial nature of asparagus and the difficulty of combating the pathogen in the soil without damaging the plant. Allelopathy Many components operating in the soil determine whether a plant will be attacked by a root pathogen. Although Asparagus Decline is ultimately attributed to Fusarium infection, many other factors involved in the agroecosystem contribute to asparagus decline. It is now well accepted by the scientific community that there are substances in the soil that are inhibitory 13 14 to plant growth (5,40,46,51). As early as 1832, DeCandolle suggested that "soil sickness" was due to crop plant exudates (43). Since then, a large amount of scientific effort has been devoted to elucidating how root exudates and leachates from plant residues affect plant growth, interference between plant populations, and the microbial populations including plant pathogens in the agroecosystem. Schroth and Hildebrand (46) observed: "Plant exudates directly affect the pathogens by inducing their germination, contributing to nutritional status prior to penetration or by inhibiting their saphrophytic and pathogenic activities. The pathogens are affected indirectly by competition and antibiosis by the root micro— flora whose activities also are mediated by exudates". This biochemical interaction that plants exert on their environment was termed "allelopathy" by Molisch in 1937. Allelopathy is defined as "any direct or indirect harmful affect by one plant (including microorganisms) on another through the production of chemical compounds that escape into the environment" (42). E.L. Rice further clarified this definition by stating "the effect (of allelopathy) depends on a chemical being added to the environment" (43). Allelopathy differs from competition; competition is defined as "the removal or reduction of some factor from the environment that is required by some other plant sharing the habitat (43)." Although the term allelopathy is rarely used in plant pathology literature, 15 development of morphogenesis of pathogens, antagonism of pathogens by non-host organisms, development of disease symptoms, host-plant resistance and promotion of infection all appear to involve allelochemicals (5,42). Allelopathic effects appear to be especially important in natural communities dominated by a single species (58). Since essentially all of the agriculture in the United States is comprised of large monocultures of plant species, it is not suprising that allelopathy may function in these agroecosystems. However, only a few researchers have examined this phenomena in any great detail in relationship to microorganisms, in particular plant pathogens. In an examination of the peach tree decline and replant problem, Chandler and Daniell (6) found peach seedlings grown in old peach soil and peach soil leachates were more susceptible to infection by Pseudomonas syringae than seedlings grown in the control soil or soil from a pecan orchard. They postulated that toxins from dead peach roots may predispose new trees to bacterial canker and thus contribute to peach decline. Patrick identified amygdalin, a cyanogenic glycoside in peach roots, as the source of toxic substances present in the soil (38). In the presence of enzymes provided by the microbial population in the soil, amygdalin is cleaved in two places and hydrogen cyanide (HCN) and benzaldehyde are produced. He demonstrated that peach trees are susceptible to damage by these compounds while other Prunus species such as apricot are less affected. Patrick and Koch (39) reported that exposure of tobacco 16 plants to leachates obtained from decomposing rye and timothy residues increased the susceptibility of tobacco to black root rot caused by ThielaviOpsis basicola (Beck. & Br.) Ferraris. Using 16 different tobacco varieties ranging from susceptible to resistant to black root rot and 6 different isolates of T. basicola, they showed that exposure to the leachates broke down the resistance to the pathogen in even the most highly resis- tant varieties. They felt the leachates were damaging the tobacco roots and,therefore, making plants more susceptible for infection and colonization by the fungus. They hypothesized toxins produced by rye or timothy accounted for the breakdown of resistance in the field since rye and timothy were often used in rotation with tobacco. They concluded these toxins may be an important "host-conditioning" factor in the disease syndrome. Kommedahl and Ohman (26) reported that Agropyron repens (L.) Beauv. (quackgrass) produces a toxin that predisposes Medicago sativa L. (alfalfa) seedlings to infection by root pathogens. Their research also indicated nitrogen overcame some of the deleterious effects of the quackgrass for cats but not soybean and the quackgrass residues harbored fungi pathogenic to alfalfa (26,27). Their evidence for toxin production from quackgrass was 1) water extracts from rhizomes decreased germination in vitro of several indicator species, and 2) when indicators were grown in fields previously infested with quackgrass, a decrease in growth height occurred when compared to the same species grown on similar soil lacking quackgrass. Yields of cats and soybean were also decreased to some extent. No attempt was made to show 17 whether there was any interaction of toxic extracts with pathoge- nicity of fungal species on indicator species and no statistics were performed for the data. They felt the toxic products were still a factor in disease development and their presence should not be ignored in the overall disease etiology. Toussoun and Patrick reported toxic products of decomposing residues of rye, barley, broccoli, and broad bean greatly enhanced the pathogenesis ole. solani (Mart.) Sacc. f. sp. phaseoli (Burk) Snyder & Hansen on beans (55). Disease enhancement, measured by lesion development on bean stems, was greatest using toxic extracts obtained during the early stages (less than one month) of decay of the residues. They postulated this enhancement was due to an additive effect of the extract and the pathogen: the extract was preconditioning the roots to fungal invasion. They noted that root rots are not necessarily caused by specific pathogens and hypothesized organisms ordinarily causing little damage might be able to "become more aggressive" if conditions were favorable for their development. Other experiments showed these toxins had a direct effect on the host cells, altering the cell permeability. They concluded the resulting increased exudation of ninhydrin— positive compounds and other substances were readily available to organisms in the infection court and were mainly responsible for predisposing the host to infection by pathogenic organisms. Asparagus as an Allelopathic Plant In 1970, VanBakel and Kerstens observed that soils used for asparagus production cannot be used again because of "soil l8 sickness", but, like others before them, attributed the problem to Fusarium infestation (57). In 1972 a group of Japanese researchers (24) isolated a substance from etiolated asparagus tissue extractable by ether that inhibited lettuce, rice, rape, radish, carrot, and barnyard grass root growth in petri dish assays. Purification and identifications of this substance indicated that the compound was 1,2-dithiolane-4-carboxylic acid, colloquially called asparagusic acid. This acid inhibited root growth of the aformentioned indicator species at 4 to 6.67 x 10‘7 M. concentrations from 6.67 X 10- The first report of possible allelopathic effects of asparagus was published in 1977 by Laufer and Garrison (28). Unsterilized soil amended with 4 g of asparagus root tissue delayed asparagus seed emergence for 11 days and strikingly inhibited the emergence of weed species. Crowns were less inhibitory and fern tissue not inhibitory in the same test. Water extracts of root tissue were also inhibitory to germin- ation of asparagus seed in petri dish assays. Their data suggested asparagus might be an allelopathic plant. During the same year asparagus root growth was found to be inhibited when grown in the presence of dried, ground, asparagus root tissue in greenhouse studies (10). Shafer and Garrison followed up this piece of evidence with more comprehensive studies on seedling emergence in the presence of dried asparagus root tissue (47). Two, 4 or 6 g amounts of dried, ground asparagus tissue was allowed to "decompose" in soil for O, 28, 50, and 90 days, then seedling emergence of lettuce, tomato, and asparagus was l9 assayed in these soils. Possible electrical conductivity and pH confounding factors were also checked. The highest rate of dried root tissue continued to delay emergence after 90 days of decomposition. All rates delayed emergence at 0 to 28 days of decomposition. They concluded that if allelopathic substances were being produced, they were inactivated with time in the soil. Laufer and Garrison also showed water extracts from dried root tissue contained substances of MW less than 1000 that were inhibitory to lettuce and seed radicle elongation but did not affect germination (29). Putnam et al. has since established that inhibitors present in water extracts are water soluble and chloroform insoluble (41). To date, the most comprehensive report of allelopathic properties attributed to asparagus was published by Yang (61). He used water extracts of dried plant tissue from both field grown and tissue cultured plants in germination and seedling development assays. Although germination of asparagus seed was delayed in the presence of extract, germination after 8 days was not significantly different from that of the distilled water controls. However, root length was significantly reduced when compared to the controls. This was true with stem, crown and root extract of both field grown and tissue cultured plant extracts. The root extract was most inhibitory but crown and stem extracts also affected growth to a lesser extent. Shoot length was inhibited by both crown and root extracts but not by stem extract. The root extract was most inhibitory. Substances present in the water extract were found to be heat stable. 20 Yang's study gave good evidence that toxic substances important in inhibition of seedling development could be extracted from asparagus plant tissues. He suggested these autotoxins may play a role in the decline problem of asparagus. He also showed that this toxic effect of the water extracts could not be diminished by adding charcoal to the preparations. He mentioned that some plant selections obtained from breeding lines in Washington were productive in old asparagus fields and suggested that varietal selection was probably the most viable way to overcome the replant problem of asparagus. In summary, not much has been done about the replant problem in asparagus since it was first reported by Hanna in 1947. Although the problem is ultimately attributed to Fusarium infestation of asparagus fields, many other factors including root exudates of plants or allelochemicals present in the agroecosystem could be contributing to the phenomenon of Asparagus Decline. In this thesis, there are three main objectives: First, to determine if there are any interactions of the reported allelopathic substances of asparagus with Fusarium spp. pathogenic to asparagus. Second, to further purify allelopathic compounds from asparagus root tissues, since these compounds may play a role in asparagus decline. And third, investigate if allelopathic compounds produce changes in the rhizosphere around the asparagus roots, consequently shifting the ecological balance of the soil and giving some competative advantage to other microorganisms in the sandy soil — specifically the asparagus pathogens, F. oxysporum f. sp. asparagi, and F. moniliforme. CHAPTER I Asparagus Tissue and Fusarium spp. interaction on the Incidence and Severity of Root and Crown Rot of Asparagus Although it is recognized that soil amendment studies are not complete proof of allelopathy, many researchers have used this method to help substantiate the role of allelopathy in plant disease development. In this section, greenhouse studies using soil amended with the different dried asparagus tissues in combination with isolates of the two Fusarium pathogens,_F. oxysporum f. Sp. asparagi and F. moniliforme were conducted a) to determine if the incidence or severity of Fusarium root and crown rot would be increased under these conditions and, b) if the phenomenon was caused by an interaction or an additive effect between plant tissue and fungal species. MATERIALS AND METHODS Preparation of Inoculum Millet seeds [Setaria italica (L.) Beauv.] (250 g) and distilled water (100 ml) were placed in a 1 liter flask, autoclaved 1 hour, shaken to loosen the seed from sides of the flask, then left to cool overnight at room temperature. The millet seeds were reautoclaved the next day for 1 hour, allowed to cool, then inoculated with a 4 mm diameter plug of actively growing mycelium of F. moniliforme or F. oxysporum f. Sp. asparagi. The cultures were grown at 260C and each flask was shaken daily to facilitate mycelial Spread throughout the millet seeds. The cultures were harvested after 2 wks and 21 22 to cool, then inoculated with a 4 mm diameter plug of actively growing mycelium Of.E° moniliforme or F. oxysporum f. Sp. asparagi. The cultures were grown at 260C and each flask was shaken daily to facilitate mycelial spread throughout the millet seeds. The cultures were harvested after 2 wks and allowed to air dry and then stored in paper bags at 260C until ready for use in soil amendment studies. Preparation of Asparagus Plant Material A commercial asparagus field in Oceana County, MI, was excavated and 'Martha Washington' asparagus plants were collected, washed, and separated into living roots and rhizomes. All dead plant material was discarded. The roots and rhizomes were oven—dried (50°C) and ground in a Wiley mill. Asparagus ferns were clipped from actively growing plants in the greenhouse and prepared in the same manner. All dried plant tissues were sterilized with prOpylene dioxide (58). Containers with sterilized tissue were allowed to exhaust under a hood for 24 hr to dissipate all propylene dioxide. Dried tissues were stored in plastic bags at -2000 until used. In order to test for Fusarium Sp. contamination, 1 g of dried tissue was spread evenly over the surface of a 9 cm petri plate containing Komada's medium. After 1 week, plates were assessed for fungal colonies growing on the media. Interaction of Fusarium spp. and Dried Asparagus Plant Tissues with ASparaguS Seedlings The effect of combinations of the two Fusarium spp. and the dried root, rhizome and fern tissue was tested on asparagus seedlings. Hybrid asparagus seedlings "UC 147", 8—wk-old, 23 were grown in steamed soil (sand:greenhouse soil, 2:1 v:v) containing the following: 1).3' moniliforme, 2) F. moniliforme and root tissue, 3).E' moniliforme and rhizome tissue, 4) F. oxysporum f. Sp. asparagi, 5) F. oxysporum f. Sp. asparagi and root tissue, 6 ).F. oxysporum f. Sp. asparagi and rhizome tissue, 7)both Fusarium isolates together, 8) both isolates and root tissue, 9) both isolates and rhizome tissue, 10) root tissue, 11) rhizome tissue, and 12) a sterilized millet seed control. In a second experiment, fern tissue was used in place of root or rhizome tissue. All other treatments were the same as in the first experiment. In each experiment, infested millet seed inoculum was incorporated into soil at 8 g millet seed inoculum/4-in. pot. When both isolates were incorporated, the inoculum was applied at 4 g. Dried plant tissues were incorporated into soil at 50 g (8% of soil weight). The inoculated seedlings were placed at random on a green— house bench and watered daily. The plants were fertilized twice with Peters fertilizer 20:20:20 during the time they were in the greenhouse. After 8 wk, the plants were harvested and evaluated visually for root and rhizome rot separately using a scale of 0-5. The data were subjected to analysis of variance and Duncan's Multiple Range Test. The effect of the two Fusarium spp. in combination with different amounts of root tissue on asparagus seedling growth was tested. A 4 X 3 factorial experiment was conducted using 0, 5, 10, 20 g root tissue with either no fungus or in combination with F. moniliforme and F. oxysporum f. Sp. asparagi. 24 were grown in steamed soil (sand:greenhouse soil, 2:1 v:v) containing the following: 1) F. moniliforme, 2) F. moniliforme and root tissue, 3) F. moniliforme and rhizome tissue, 4) F. oxysporum f. Sp. asparagi, 5) F. oxysporum f. Sp. asparagi and root tissue, 6 )‘F. oxysporum f. Sp. asparagi and rhizome tissue, 7) both Fusarium isolates together, 8) both isolates and root tissue, 9) both isolates and rhizome tissue, 10) root tissue, 11) rhizome tissue, and 12) a sterilized millet seed control. In a second experiment, fern tissue was used in place of root or rhizome tissue. All other treatments were the same as in the first experiment. In each experiment, infested millet seed inoculum was incorporated into soil at 8 g millet seed inoculum/4-in. pot. When both isolates were incorporated, the inoculum was applied at 4 g. Dried plant tissues were incorporated into soil at 50 g (8% of soil weight). The inoculated seedlings were placed at random on a green— house bench and watered daily. The plants were fertilized twice with Peters fertilizer 20:20:20 during the time they were in the greenhouse. After 8 wk, the plants were harvested and evaluated visually for root and rhizome rot separately using a scale of 0-5. The data were subjected to analysis of variance and Duncan's Multiple Range Test. The effect of the two Fusarium Spp. in combination with different amounts of root tissue on asparagus seedling growth was tested. A 4 X 3 factorial experiment was conducted using 0, 5, 10, 20 g root tissue with either no fungus or in combination with F. moniliforme and F. oxysporum f. Sp. asparagi. 25 There were 6 plants/treatment. Inoculum was applied at 8 g. Plants were grown as before, harvested after 8 wk and evaluated for root and rhizome rot, fresh weight, and dry weight. The data were subjected to an analysis of variance and Duncan's Multiple Range Test. RESULTS Sterilized plant material Showed some residual Fusarium infestation. Eight of the 10 plates Showed some fungal growth present with an average of 2.9 colonies/ plate, standard deviation of 2.45, and a range of 0-8 colonies/plate. None of these colonies were checked for pathogenicity. We considered this amount of residual colonization of the plant tissue by Fusarium spp. too low to contribute to infection caused by our pathogens in our experiments. In the first experiment, dry weights of seedlings treated with root tissue alone or with root tissue and Fusarium spp. were Significantly lower (F;0.05) than dry weights of controls (Table 1). Neither pathogen, rhizome tissue alone, or Fusarium oxysporum f. sp asparagi +.E' moniliforme + rhizome tissue Significantly decreased dry weights as compared to the controls. However, root rot rating of seedlings treated with F. oxysporum f. Sp. f. Sp. asparagi or F. moniliforme or asparagus plant tissues were significantlly greater than those of the controls (Figure 4 and 5). Root rot was greatest in treatments of root tissue combined witth. moniliforme or F. oxysporum f. Sp. asparagi. Analysis of data was done using data transformed by square root. 26 emowo A. was Sowmwa mum woos woe wmdwsm How mmwmommcm moomwwsmm awomaom sad: oosowsmdwosm ow acmmowds mob. SSS newoo mmbmwmmsm awmmco. eHmmdw Zowm " wooe mmmmozm H H me e um& amHmma made wag 8mm admmma wade woe wdm>dez mew. Amv wbeHzm Amv w>eHzm Amv w>eHzm zose m.mm as 0.0 m o.mm me u.m e m.sm s k.o e @o m.m# a H.m o o.wm md a.m w 0.0m moo w.m a oz m.o< a m.m o o.me m s.m w H.ea do m.m a we + w: H.0e o H.w d o.mw mo m.m o m.om a w.w no H w: u.w. BOSMHHwowSo. so u w. oxwmwowsa w. ma. mmwmemmw. m Uo% Sowmwa u Bow: 0% m wowwwomawosm\awoma5osa. w zomsm swasoca m Hoaaoo as oossos mew mMmSHwHomSaHM awwwooosa ma w n 0.0m Woe USSomS.m zzwawwwo wmbmo eoma. a weed woe Hmawsm momwo“ o n so HOOd Hoe ow wwwuoSo mwmoowowmdwos. A H mm& wooa wow ow was won ow wwsw maeome HS aso owwnoSo. w u mos oooa woe ow vooswsosa mdwomstm HS HSHN05o dammSom. w u 4mm HOOd wow ow mossy ow Mme 0% arm wwwnoSo. a u mommaoo arm: QmX oooa woe ow moms: ow mom 0% arm HSHNoBm. m u Some: om arm meSd. 27 Figure 4. Root rot of asparagus caused by a combination of F. oxysporum f. sp. asparagi and asparagus dried tissue‘lcrown residue = dried rhizome tissue, root residue = dried root tissue). —m Figure 5. Root rot of asparagus caused by a combination of t I I l m ,- r‘ /‘ KNLJ / / ‘ v / . l, / I c-yipowm 9 lullrnduc 28 The AOV indicated there was no significant interaction of the Fusarium X tissue combination at the F=0.05 level (Table 2). This indicates an additive effect of the Fusarium spp. and root tissue. In the second experiment, there was no Significant re- duction in dry weight in the treatment with fern tissue alone (Table 3). Seedling dry weight was decreased Significantly by the addition of F. oxysporum f. Sp. asparagi and F. moniliforme and even more so when the pathogens were incorporated into the soil together. However, the addition of fern tissue in combination with F. oxysporum f. Sp. asparagi did not decrease dry weight to any greater extent than when F. oxysporum f. Sp. asparagi was incorporated into the soil alone. When fern tissue was incorporated into the soil in combination with F. moniliforme, dry weight decreased more than when F. moniliforme alone was present. Fern tissue alone caused no Significant increase in root rot rating compared to controls (Table 3). Root rot ratings did increase when either pathogen singly or in combination was incorporated into the soil. The addition of fern tissue in combination with F. moniliforme caused a greater increase in root rot than when F. moniliforme alone or fern tissue alone was incorporated into the soil. However, there was less root rot of seedlings in those treatments where fern tissue F. oxysporum f. Sp. asparagi were present than when F. oxysporum f. Sp. asparagi alone was present. In the third experiment, when asparagus seedlings were 29 Table 2. Analysis of variance of dry weight of 3 month old asparagus seedlings treated with Fusarium spp. and dried asparagus root or rhizome tissue. 1 Sum of Degree 3f Mean ' Level of Treatment Squares Freedom Square Statistic Significance of F Value Fusarium spp.3 0.0018 1 0.0018 0.014 0.91 Root or rhizome 5.237 2 2.613 20.564 0.0005 rhizome tissue Fusarium X 0.336 2 0.169 1.321 0.282 tissue interaction Error 3.81 30 0.127 1 Data analysis on data transformed by square root. 2 Treatments arranged in a 3 X 4 Factorial design with 6 replications/treatment. 3 Fusarium spp. were F. oxysporum f. Sp. asparagi or F. mon1I1Iorme o 30 Table 3. Dry weight and root rot rating for asparagus seedlings treated with combinations of Fusarium spp. and dried asparagus fern tissue. Treatment Dry Weight3 Root Rot5 (gm) Rating FMl + fern tissue .03 a4 3.3 f FM + F02 + fern tissue .15 ab 2.3 de PM + F0 .22 abc 4-5 g F0 + fern tissue .42 cd 1.6 cd F0 .51 de 2.7 ef FM .65 e 1.0 bc Fern tissue 1.03 f 0.3 ab Control 1.12 f 0.0 a 1PM eig. moniliforme. 2F0 =‘F. oxysporum f. Sp. asparagi. 3Dry weight = mean of 6 plant replications/treatment. 4Means without a letter in common are Significantly different at 3': 0.05 for Duncan's Multiple Range Test. Means were also analyzed using orthoganol contrasts. 5Root rot rating Scale: 0 = no root rot or rhizome discoloration, 1 = 25% root rot or few red or pink streaks in the rhizome, 2 = 50% root rot or prominent streaking in rhizome tissue, 3 = 75% root rot or death of 25% of the rhizome, 4 greater than 75% root rot or death of 50% of the rhizome, 5 death of the plant. 31 planted into soil containing combinations of Fusarium Spp. and different levels of root tissue, analysis of variance of the 3 X 4 factorial experiment indicated there was a Significant interaction of dried root tissue and Fusarium spp. on fresh weight and dry weight of 3-month old asparagus seedlings (Tables 4 and 5). When the 10 g or 20 g level of root tissues were incorporated into the soil in combination with F. moniliforme, dry weight, as well as fresh weight was decreased Significantly when compared to the control or to the treatment using F. moniliforme alone (Tables 6 and 7). Curiously, when_F. oxysporum f. Sp. asparagi was incorporated into the soil alone, some increase in dry weight and fresh weight were noted. However, as increasing levels of dried root tissue were incorporated into the soil with F. oxysporum f. Sp. asparagi, a progressive decrease in dry weight was observed. Root rot ratings indicated there was little disease evident in plants treated with either pathogen (Table 8). However, visual assessment of root rot increased as levels of tissue incorporated into the soil increased. Root rot rating also increased when dried tissue was incorporated into the soil in combination with either pathogen. DISCUSSION These experiments indicate that severity of Fusarium root and crown rot caused by F. oxysporum f. Sp. asparagi and F. moniliforme is increased in the presence of dried plant tissues. 32 Table 4. Dry weights of 3 month old asparagus seedlings treated with varying levels of dried asparagus root tissue alone or in combination with F. oxysporum f. Sp. asparagi or f. moniliforme. _ Tissue Weight (g) 10 Fusarium Spp.l 0 5 20 No Fusarium 1.27 ed2 1.25 bcd 1.14 bcd 0.75 a F0 2.16 e 2.13 e 1.26 bcd 0.87 abc FM 1.53 d 2.38 e 0.43 a 0.73 a lFusarium treatments: No Fusarium, F05E° oxysporum f. Sp. asparagi, FM = F. moniliforme. No Fusarium control was sterilized miIIet seed incarporated into soil. 2Means with out a letter in common are significantly different at P=0.05 for Duncan's Multiple Range Test. 33 Table 5. Fresh weight of 3 month old asparagus seedlings treated with varying levels of dried asparagus root tissue alone or in combination with F. oxysporum f. Sp. asparagi or F. moniliforme. Tissue Weight (g) Fusarium Spp.l 0 5 10 20 No Fusarium 4.82 e2 5.71 cd 5.75 cd 2.03 a F0 6.27 g 5.97 f 4.07 de 5.15 0 FM 4.62 de 6.72 g 2.20 ab 2.63 ab lFusarium treatments: no Fusarium, FOSF. oxysporum f. Sp. asparagi, FM = F. moniliforme. No Fusarium control was sterilized miIIet seed incorporatedIInto soil. 2Means with out a letter in common are Significantly different at F=0.05 for Duncan's Multiple Range Test. 34 Table 6. Analysis of variance of fresh weight of 3 month old asparagus seedlings treated with varying levels of dried asparagus root tissue alone or in combination with F. oxysporum f. Sp. asparagi or F. moniliforme. Sum of Degree 3f Mean F Level of Treatment squares freedom square statistic Significance of F Value Fusarium spp.2 20.5 2 10.15 6.80 0.002 Root tissue3 107.47 3 35.82 23.99